Combination vaccine for enhancing immunity against brucellosis

ABSTRACT

A vaccine comprising a combination of Brucella “A” and “M” outer-polysaccharides (OPSs) and “R” protein antigens for enhancing immunity against brucellosis is disclosed. The OPS may be obtained from different strains or species of Brucellae (i.e. combining OPS extracted from different bacteria expressing “A” or “M” OPS, or combining OPS and OPS-protein complexes extracted from different bacteria). The OPS or OPS-protein complexes may also be obtained from a single strain expressing more than one OPS (e.g. from  B. suis  strain 145 which expresses “A”, “M” and possibly other OPSs). The vaccine according to the present invention overcomes the limitation of previously discovered  B. abortus  “A” OPS which only protects against species and strains of Brucella that had “A” OPS but not against others with different OPS.

FIELD OF INVENTION

[0001] The present invention relates to a vaccine, comprising acombination of bacterial components derived either from differentspecies of Brucellae, or one strain expressing different components,that enhances immunity against brucellosis. The vaccine formulations areapplicable for one or more cross-reactive bacteria thereof.

BACKGROUND OF THE INVENTION

[0002] Brucellosis is a debilitating disease that can cause abortionsand weight loss in animals as well as undulating fevers, night sweats,incapacitation and arthritis in humans. It is very hardy toenvironmental factors, easily aerosolized and infectious through skinabrasions, ingestion and the pulmonary route. It is difficult to treatwith antibiotics and often persists as a life-long infection.Brucellosis is a disease endemic to most countries, especiallyunder-developed nations where Brucella species infect 0.1 to 10% of thelivestock such as cattle, swine, sheep, goats, and camels. A zoonoticdisease, these also infect other domestic animals such as dogs andpoultry, wildlife such as bison, caribou and wolves and marine mammalssuch as whales and dolphins. People are especially vulnerable toinfection either through handling infected products or ingestingcontaminated foods.

[0003] Up-to-date, effective treatment against brucellosis for animals,including humans, has been limited. For humans, administering high dosesof combination antibiotics, for example doxycycline with rifampin overlong periods, has been found to be effective to clear the disease, butnon-compliance and relapses are common. For animals, the cost andlimited effectiveness of antibiotic treatments often lead to thedecision of either no treatment or elimination of the infected animaland its associated herd.

[0004] The most preferred type of disease management is to avoidinfection and to reduce the incidence and spread of the disease byvaccination. For livestock, namely cattle, at present vaccinationconsists of using an attenuated (weakened) vaccine strain such asBrucella abortus strain 19. Although it is one of the best vaccines forcattle, it does have limitations in that the vaccine does not giveabsolute protection and there is about a 20% failure rate, results fromserological tests can be confusing for a positive serology may be causedby vaccination, infection, or vaccination with subsequent infection, thevaccine although tolerated by cattle is pathogenic for humans, and onoccasion the vaccine does revert to a “wild” or virulent form.

[0005] For humans, there existed a French vaccine that consisted of aphenol insoluble residue. However, this vaccine has been discontinued asit was found that the residue caused a high rate of reactogenicity (inone study, a large percentage of the vaccine recipients developedswollen lymph glands and granuloma at the site of injection) andhyper-sensitivity (vaccinates that touched killed Brucella preparationspresented symptoms of anaphylactic shock).

[0006] Recently, the Applicant has discovered a new vaccine thatprotected animals (e.g. mice, guinea pigs and swine) from brucellosisand which may upon further development be suitable for protectinghumans. The vaccine is as described in U.S. Pat. No. 5,951,987 which isherein incorporated by reference. The vaccine consists of anouter-polysaccharide (OPS) isolated from Brucella such as Brucellaabortus. The vaccine protected animals from different strains andspecies of Brucella tested (e.g. B. abortus 30, B. abortus 2308 and B.suis biovar 1) as well as infections from Francisella tularensis livevaccine strain (LVS) which causes tularemia in mice. This gave evidencethat the vaccine would likely offer effective protection againstinfections from a broad spectrum of Brucella species and cross-reactivebacteria. However, it has subsequently been found otherwise. Althoughthe B. abortus OPS vaccine was effective in offering animals protectionfrom brucellosis, it did so only against species and strains thatresembled B. abortus in serology (i.e. had “A” OPS antigens). It did notappear to be effective against species and strains that resembled B.melitensis in serology (i.e. had the “M” or “A&M” OPS antigens). Hence,there still remains a need for a vaccine which is effective againstinfections from a wide spectrum of Brucella species.

SUMMARY OF THE INVENTION

[0007] In accordance with one aspect of the present invention, there isprovided a vaccine comprising a combination of Brucella “A” and “M”outer-polysaccharides and “R” protein antigens. Theouter-polysaccharides may be obtained from the same or different speciesof Brucellae. The most preferred source of OPS is derived from Brucellabut a logical extension of this finding is to use bacterial speciescross-reactive thereof.

[0008] The combination may be obtained from combining “A”outer-polysaccharides extracted from Brucella species selected from thegroup consisting of B. abortus biovar 1, B. abortus biovar 2, B. abortusbiovar 3, B. abortus biovar 6, B. melitensis biovar 2, B. suis biovar 1,B. suis biovar 2, B. suis biovar 3, B. neotomae and B. maris; “M”outer-polysaccharide extracted from Brucella species selected from thegroup consisting of B. abortus biovar 4, B. abortus biovar 5, B. abortusbiovar 9, B. melitensis biovar 1, B. suis biovar 5; and “R” corepolysaccharide and proteins extracted from Brucella species selectedfrom the group consisting of B. ovis and B. canis.

[0009] Alternatively, the combination may be obtained by combining “AM”outer-polysaccharides extracted from Brucella species selected from thegroup consisting of B. abortus biovar 7, B. melitensis biovar 3 and B.suis biovar 4 (note: B. suis 145 biovar 4 is used in the present patentsubmission), and “R” core polysaccharide and protein extracted fromBrucella species selected from the group consisting of B. ovis and B.canis.

[0010] In accordance with another aspect of the present invention, thereis provided a vaccine comprising a combination of Brucellaouter-polysaccharides containing the “A” and “M” antigens and a Brucellaouter-polysaccharide-protein complex.

[0011] In this case, the combination may be obtained by combining “A”outer-polysaccharide purified from Brucella species selected from thegroup consisting of B. abortus biovar 1, B. abortus biovar 2, B. abortusbiovar 3, B. abortus biovar 6, B. melitensis biovar 2, B. suis biovar 1,B. suis biovar 2, B. suis biovar 3, B. neotomae and B. maris; “M”outer-polysaccharide purified from Brucella species selected from thegroup consisting of B. abortus biovar 4, B. abortus biovar 5, B. abortusbiovar 9, B. melitensis biovar 1, B. suis biovar 5; and anouter-polysaccharide-protein complex selected from the group consistingof outer-polysaccharide and Brucella membrane proteins,outer-polysaccharide and Brucella surface proteins, outer-polysaccharideand Brucella surface enzymes and outer-polysaccharide and Brucellacytoplasmic proteins.

[0012] Alternatively, the vaccine may be obtained by combining “AM”outer-polysaccharides extracted from Brucella species selected from thegroup consisting of B. abortus biovar 7, B. melitensis biovar 3 and B.suis biovar 4, and an outer-polysaccharide having a protein selectedfrom the group consisting of Brucella membrane proteins, Brucellasurface proteins, Brucella surface enzymes and Brucella cytoplasmicproteins.

[0013] The vaccine may consist of 1 ng to 10 ug, preferably 1 ug, ofeach of the OPS forming the combination for vaccination of mice weighingabout 20 grams.

[0014] The vaccine is effective as a prophylactic treatment frominfection against a wide range of Brucella species namely B. abortus, B.melitensis and B. suis. By logical extension the vaccine is likely to beeffective for the prevention of brucellosis from B. ovis, B. canis, B.neotomae and B. maris. Animal studies support its use as a vaccine forlivestock, and with further development possibly as a vaccine forhumans. It is most effective by intra-peritoneal, sub-cutaneous andintramuscular administration. It is least effective when givenintra-nasally. The vaccine works best against the most virulent speciesand strains of Brucella, most of the healthy vaccinates having nobacteria in their spleens or having a million fold less bacteria thancontrols. The vaccine works, but is less effective where it is notneeded, or in mice given Brucella species and strains of low virulence.

[0015] Serum or white blood cells of mammals vaccinated with the vaccinein accordance with the present invention prevented brucellosis inrecipient animals. Protection is long term (i.e. at least several weeks)but unlike other vaccines it is also protective in the short term (i.e.protective in 1 day or less).

DETAILED DESCRIPTION OF THE INVENTION

[0016] Brucella species can be classified by their different type ofouter-polysaccharides (OPS). These serological types are those havingthe “A” OPS, the “M” OPS and those lacking OPS or the “R” group (i.e.antigens are predominantly protein with some antigenicity being the“core” polysaccharides attached to the lipopolysaccharide, or LPS,lacking OPS), wherein “A”, “M” and “R” stands for the type of antigens.Some species also express more than one antigen, for example somestrains of B. suis (biovar 4) which express both the “A” and “M”antigens, and some are capable of changing their antigens within thesame host (personal communications, Dr. G. G. Schurig, 1997). Thevarious types of OPS are very similar in chemical structures. They aregenerally made of identical sugars, which are linked differently.Because of the similarity between the OPS and the considerablecross-reaction between the “A” and “M” OPS of Brucella, one would expecta single OPS vaccine, i.e. a vaccine consisting of one type of OPS, tobe effective against a wide range of Brucella species. This, however,was found to be only partially true. The Applicant discovered that acombination or “cocktail” vaccine, i.e. a vaccine having a combinationof the different OPS, is much more effective than the single OPS vaccinein providing protection against a wide spectrum of Brucella species.

[0017] To be most effective, the cocktail or combination vaccine shouldinclude a range of OPS, for example both “A” and “M” OPS, and “R”antigens. As it is likely that one or more OPS, other than “A” and “M”,are also produced (Vizcaino, N., Cloeckaert, A., Zygmunt, M. S., andFermandez-Lago, L. 1999. Molecular characterization of a Brucellaspecies large DNA fragment deleted in Brucella abortus strains: evidencefor a locus involved in the synthesis of a polysaccharide. Infection andImmunity, 67: 2700-2712), the inclusion of these would likely offergreater protection.

[0018] The cocktail or combination vaccine may be replaced by anouter-polysaccharide-protein complex. Brucellae can attach proteins (the“R” antigen, which can also be extracted from cells that do not expressthe “A” or “M” antigen, have colonies rough in appearance and expressmainly proteins) to OPS. OPS and OPS-protein complex can be separated(e.g. with 0.2 M trichloroacetic acid, OPS remains soluble, OPS-proteinprecipitates).

[0019] Outer-polysaccharides containing the “A” antigen can be obtainedfrom B. abortus biovar 1, B. abortus biovar 2, B. abortus biovar 3, B.abortus biovar 6, B. melitensis biovar 2, B. suis biovar 1, B. suisbiovar 2, B. suis biovar 3, B. neotomae and B. maris.

[0020] Outer polysaccharides containing the “M” antigen can be extractedfrom B. abortus biovar 4, B. abortus biovar 5, B. abortus biovar 9, B.melitensis biovar 1, B. suis biovar 5.

[0021] Proteins, core polysaccharides and short chainedouter-polysaccharides comprising the “R” antigen can be purified from B.ovis and B. canis.

[0022] Outer-polysaccharides containing both the “A” and “M” antigenscan be purified from B. abortus biovar 7, B. melitensis biovar 3 and B.suis biovar 4.

[0023] Suitable proteins for the “R” component are Brucella proteins.Preferably, they are outer-membrane proteins (opm) such as opm1, opm2and opm3, lipoprotein linked to cell wall, porin (a protein that allowsions or metabolites through the membrane), A5 on the cell surface,surface proteins such as a, b and X, surface enzymes such as protease,Brucellin proteins and internal or cytoplasmic proteins. Other proteinsmay be mannosyltransferase, GDP-mannose 4,6 dehydratase, perosaminesynthetase, ABC-type transporter and formyl transferase. Additionalproteins such as those identified in the paper “Conservation in Brucellaspp. of seven genes involved in the biosynthesis of thelipopolysaccharide O-chain”, A. Cloeckaert, M. Grayon, J-M Verger, J-JLetesson and F. Godfroidt, 1998. 51th Annual Brucellosis Meeting,Chicago, herein incorporated by reference, can also be used.

[0024] To assess the effectiveness of the vaccine, different single OPSvaccine and combination OPS vaccines were prepared and tested. The novelformulation of using combination OPS vaccine of the present invention isshown to be more effective than a single OPS vaccine formerly disclosedby the Applicant in the U.S. Pat. No. 5,951,987.

[0025] Materials and Methods

[0026] Bacterial Cultures:

[0027]B. abortus 30, B. abortus 2308, B. melitensis 16M and B. suis 145were acquired in 1989 from the Animal Diseases Research Institute(ADRI-Nepean, now the Canadian Food Inspection Agency), Nepean, Ontario,Canada. The bacteria were thawed and small aliquots of the materialswere obtained and grown on Brucella agar (Difco Laboratories, Detroit)with 1.5 ppm crystal violet at 37° C., 5% CO₂, 90% humidity, for 1 week.A loopful (about a billion cells) of the culture were placed in vialscontaining 1 ml of sterile Brucella broth with 15% glycerol and thevials were frozen at −70° C. As required, vials containing B. melitensis16M, B. suis 145, B. abortus 30 and 2308 were thawed and subculturedonto, for example, Brucella agar and incubated to provide cells forProtect Beads™ storage, or into Brucella broth and incubated to providecells which were later used in the infectivity experiments.

[0028] Representative vials of the bacteria were thawed and used toinoculate Brucella agar slants (2 cc agar in a 5 cc vial). The cultureswere verified on May 10, 1999 by the National Veterinary ServicesLaboratories (Ames, Iowa), which confirmed that the vials containing B.abortus 30 or B. abortus 2308 belonged to B. abortus biovar 1 (“A”antigen predominant), B. melitensis 16M belonged to B. melitensis biovar1 (“M” antigen predominant), B. suis 145 belonged to the atypical B.suis biovar 4 (has both “A” and “M” antigens).

[0029] The bacteria used to cause Brucella infection were prepared byinoculating bacteria such as B. melitensis 16M into Brucella broth(Difco Laboratories, Detroit), grown overnight, then 0.010 ml wastransferred to 100 ml Brucella broth and used within 18 hours ofincubation. This method revealed more effective in providing bacteriawith high virulence than the conventional method of simply thawing afrozen stock of bacteria and determining the colony forming unit (CFU),or adding the thawed stock to prewarmed Brucella broth and incubatingfor 2 hours at 37° C., 5% CO₂ and 90% humidity.

[0030] The best way to ensure virulence of the bacterium (e.g. B.melitensis 16M) was to passage it through sets of 5 mice. Hence aculture that appeared to have lost its virulence, would be given to eachof 5 mice by intra-peritoneal injection. In this case, each mouse wasgiven 5×10⁴ to 5×10⁵ bacteria/0.1 ml sterile saline. After 1 week, themice were sacrificed, their spleens weighed and crushed in saline, andserially diluted and plated on Brucella agar with 1.5 ppm crystal violetand incubated for 2 hours at 37° C., 5% CO₂ and 90% humidity and thecolony forming unit was determined. Bacteria on these plates that werefrom the mouse with the largest spleen size and greatest bacterialnumber in the spleen was selected and these were used to infect another5 mice and the selection was repeated. Within 3 passages, the bacteriumhad exceptional virulence (caused large spleens, 3-5 fold larger thannormal, and high bacterial numbers, about 3 million bacteria perspleen). These bacteria were used only for a few experiments as it wasviewed that passage through an animal could introduce other unknownvariables.

[0031] Vaccine Preparation:

[0032] a) Preparation of “A” OPS, “M” OPS or OPS-protein Vaccines

[0033] The method of preparation for some of the vaccines (e.g. “A” OPSfrom B. abortus 1119-3, “M” OPS from B. melitensis 16M) was aspreviously described (“Antigens of Brucella”, J. W. Cherwonogrodzky etal., pages 54-55, In: K. Nielsen, J. R. Duncan (ed.) 1990 AnimalBrucellosis CRC Press, Boca Raton). Briefly, each bacterium was grown onabout 90 ml of Brucella agar with 1.5 ppm crystal violet into each oftwenty 150 cm² sterile tissue culture flasks and incubated for 2 hoursat 37° C., 5% CO₂ and 90% humidity. After the incubation period, 5 ml ofsterile 3% acetic acid with 1% saline was added to each flask, a dozensmall glass beads were added, and the cells made into a suspension byrocking or lightly shaking the flask as required. The suspension wasremoved with a pipette and placed into a 250 ml centrifuge bottle.Another 5 ml of sterile acetic acid with saline in triply distilledwater was added to the flask. The flask was rocked or shaken asrequired, and the suspension was added to the previous one.

[0034] Once all twenty flasks were processed, the suspension was shakenvigorously, and stored at 4° C. for at least one week. Thereafter, itwas autoclaved at 121° C., 15 psi for 2 hours with a loosened cap. Onceautoclaved and cooled, the bottle was centrifuged at 15,000× g for 30min at 4° C. The supernatant was kept and the cells discarded.

[0035] The supernatant was neutralized with 10 M NaOH and had 2 Mtrichloroacetic acid (TCA) added to a final concentration of 0.2 M TCA.This was centrifuged (20,000× g, 30 min, 4° C.). The OPS remainedsoluble in the supernatant, the OPS-protein that was precipitated by theTCA was in the pellet. The pellet was resuspended in triply distilledwater. Both the supernatant and the redissolved pellet (kept separate)were extracted with equal volumes of liquified phenol (90% phenol with10%water) added. The mixture was magnetically stirred for 30 min at 70°C. and was chilled at 4° C. overnight. The bottom phenol layers wereremoved and centrifuged as before to remove debris or the remainingwater layer. To the phenol layers were added 5 volumes of methanol with1% sodium acetate to enhance precipitation and this was chilledovernight at 4° C. The preparations were centrifuged as before, washedtwice more with a similar volume of methanol-acetate, and the pellet wasdissolved in triply distilled water and dialyzed (1000 mw cutoff)against triply distilled water. Once dialyzed, these samples werecentrifuged to remove denatured material and lipopolysaccharide (LPS,this aggregates in distilled water while outer-polysaccharide (OPS)remains soluble). The samples were freeze-dried, weighed and kept at−70° C. until required. The resulting samples were at least 90% pure.

[0036] b) Preparation of B. suis 145 OPS Vaccine

[0037] When the previous method for OPS preparation was applied to B.suis 145, it was unsuccessful. Neither OPS nor OPS-protein precipitatedwhen methanol-acetate was added to the phenol extracts. The material wasstill present, as evidenced by OPS and OPS-protein precipitating whenthe phenol/methanol-acetate solutions were kept at −20° C. instead of 4°C. for a week, but it was clear that the procedure had to be changed toprepare OPS or OPS-protein for vaccines. (It was subsequently found thatB. suis 145 OPS is more heterogeneous in composition than the otherBrucella OPS cited, explaining the failure of the previous method, Dr.Brad Berger, unpublished results.)

[0038] In brief, B. suis 145 was grown on agar medium (Brucella agar,with or without 1.5 ppm crystal violet, trypticase soy agar, with orwithout 1.5 ppm crystal violet, gave similar results) in sterile 150 cmtissue culture flasks, at 35° C., 5% CO₂ and 90% humidity for 1 week.After this time, cells were both killed and removed by adding 5 ml of 5%phenol/1% saline, adding glass beads, and rolling or shaking the flaskto dislodge the cells, removing the cells, adding another 5 ml ofphenol-saline, rolling and shaking the flask again, and pooling cellsuspensions. From 400 flasks, about 250 grams wet weight of cells wasremoved and the final volume was about 3 liters in sixteen 250 mlcentrifuge bottles. The suspension was kept 1 week at 4° C., withoccasional shaking, to ensure release of loosely bound antigens.

[0039] After 1 week, the suspension was centrifuged (15,000× g, 20 min,4° C.), the supernatants pooled, and the cells washed with a smallvolume (40 ml per centrifuge bottle, centrifugation as before) ofphenol-saline. The liquid was added to the pooled supernatants.

[0040] To the supernatant, glacial acetic acid was added to a finalvolume of 3%. This suspension was then placed in a boiling water bathfor 2 hours. It was left to cool to room temperature for a day. The pHwas not adjusted (the low pH appears to enhance precipitation at a latermethanol stage). A one-half volume of 90% phenol (10% water) was added(the smaller volume concentrates the OPS). This was magnetically stirredon a magnetic hot-plate until the temperature rose to the mixture's“clarity” point (the phenol-water mixture was opaque, but around 65-70°C. the phenol and water dissolved into each other, causing the mixtureto clear). It was then allowed to cool at 4° C. overnight, centrifugedand the phenol layer (bottom layer, usually dark red in colour) kept. A2-week sterility check is done to ensure this phenol layer is sterileand it is then taken out of Biocontainment 3. Once in the generallaboratory area, the phenol was chilled overnight at 4° C. as was themethanol-acetate (methanol with 1% sodium acetate.3H₂O). Five volumes ofmethanol-acetate was added to the phenol layer, this was mixed on amagnetic stirrer and allowed to settle for 1-2 days. After this time,most of the liquid above the precipitate was aspirated away (the flaskis placed on ice to prevent mixing if it begins warming to roomtemperature). The remaining preparation was centrifuged as before, thesupernatant discarded, and the pellet resuspended in methanol-acetate (asaw-toothed OmniMix™ is best for blending the suspension) andcentrifuged. The pellet was then resuspended in distilled water, placedin a 1000 mw cutoff dialysis cellulose membrane, and dialyzed againstdistilled water at 4° C. (From the frothing that occurred when theoutside water was discarded during changes, it appeared thatconsiderable amount of small m.w. OPS was lost in this process, but thata large amount of larger m.w. OPS was retained by the dialysis.) Afterdialysis, the preparation was removed from the dialysis bag, andcentrifuged to remove denatured material. The pellet of denaturedmaterial was discarded, the supernatant was kept, and to the latter 2 Mtrichloroacetic acid (TCA) was added until the final concentration was0.2 M TCA. This was then centrifuged. Both the supernatant (containingOPS) and the pellet (containing OPS-protein, this was suspended indistilled water) were dialyzed against distilled water. After extensivedialysis, each preparation was centrifuged to remove denatured orparticulate material. The solutions were then aliquoted andfreeze-dried. The OPS could be further refined with enzyme digestion(though the starting material was at least 90% pure, having nodetectable A260/A280 nm absorbing nucleic acids and only about 0.6%protein) and ultra-centrifugation (120,000× g, 4° C., 3 hours) to removetrace amounts of LPS.

[0041] For the washed B. suis 145 cells noted previously, these weresuspended in 3% acetic acid and 1% saline (for every gram of cells, 5 mlof acetic acid-saline was added) and placed in a boiling water bath for2 hours with swirling to mix the suspension every half hour. The cellswere left for a day to cool to room temperature. The preparation wascentrifuged, and the supernatant kept. The cells were mixed in an equalamount (w/v) of acetic acid saline, centrifuged, and the liquid pooledwith the other. A phenol extraction was done (half a volume of phenolwas used with the cell supernatant) on this liquid as noted before. TheOPS released from the cell by boiling water was by the method notedabove.

[0042] The cells from the above were resuspended in 5 volumes of aceticacid-saline, autoclaved (121° C., 15 psi, 2 hours), cooled, centrifuged,and the liquid (as well as a washing of the cells with an equal amountof acetic acid-saline which was added) was processed as before. Thedifferent fractions noted above have been tested (1 ug/0.1 ml sterilesaline/mouse, intraperitoneal injection). All OPS fractions and allOPS-protein fractions were protective (about 10,000 fold less bacteriain their spleens than controls 1 week after challenge). The whole cellwith OPS removed, or the interphase material (between the phenol andwater layers during extraction) were not protective (indeed, theinterphase material was immuno-suppressive, causing mice to have about5-fold more bacteria in their spleens than non-vaccinated control mice).

[0043] About 4-fold more OPS was acquired if the B. suis 145, waspassaged through a mouse before being grown on agar. There was also aconsiderable amount of brown gelatinous material on the cells afterautoclaving (about 20 c.c. on 250 grams of cells, when 2 cc of this wasfreeze-dried and gave 80 mg dry weight). However, as passaging through amouse might be introducing new variables, frozen stocks kept onProtectBeads™ at −70° C. were used as the inoculum for OPS andOPS-protein production.

[0044] The strength for using B. suis 145 as a source of vaccine is thatas it expresses both “A” and “M” OPS (as well as Brucella “R” proteins)it is also likely to express other OPS. Recently another OPS has beenisolated, by a method unobvious to anyone skilled in the art. There wasan insufficient amount of this polysaccharide to characterize, otherthan it was polysaccharide, but animal studies showed that this OPS(which is in the OPS vaccine preparation) also protects mice frombrucellosis (Dr. Brad Berger, unpublished results).

[0045] As stated previously in this text, the B. suis 145 OPS orOPS-protein preparations were found to be potent vaccines that protectedmice from brucellosis. Against the most virulent strains and species ofBrucella, vaccinated mice often had no bacteria in their spleens, oronly a few (i.e. a million fold less than controls). For the latter, itwas unknown if these were simply the last traces of the challenge thatwere soon to be cleared (which is likely for their growth lag on agarmedium suggests that these are heavily damaged by the immune system ofthe host) or whether these were mutants with polysaccharides differentfrom what the mice were vaccinated against. These “survivors” wereallowed to continue growing on the agar plates previously used to assessspleen bacterial loads (35° C., 5% CO₂, 90% humidity, for an additional3 weeks), these were scraped from the plates and suspended (3.3 gramswet weight of cells was recovered) in 5% phenol, 1% saline (50 ml) andprocessed as noted above. Mice have been vaccinated with B. suis 145 OPS(as noted above, 1 ug/mouse by intraperitoneal injection), or OPSprepared from B. suis 145 bacterial “survivors” from vaccinated micethat were challenged (1 ug/mouse by i.p.), or B suis 145 OPS+B. suis 145“survivors” OPS (both 1 ug/mouse by i.p.). This study is underway but atthe time of this patent submission it has not determined if the OPS frombacterial “survivors” is equivalent to B. suis 145 OPS for protectingmice from brucellosis or whether it acts synergistically to enhancevaccine efficacy.

[0046] Mice:

[0047] Unless otherwise specified, mice were 19-21 grams at the start ofthe experiment. They were female balb/c mice obtained from CharlesRiver, St. Contance, Quebec, Canada.

[0048] Vaccination:

[0049] Mice were vaccinated intra-peritoneally (i.p) into the lowerright side of the belly, sub-cutaneously (s.c.) into the nape of skinbunched on the back, intramuscularly (i.m) into the upper left leg orintra-nasally (i.n.). The intra-nasal route required mice to beanaesthetized with Metofane™. Generally, the vaccines comprised OPS fromB. abortus 1119-3, B. melitensis 16M and/or B. suis 145 in sterilesaline as a single dose per mouse, or a combination of OPS andOPS-protein. For example, a single antigen vaccine can be 1 ug of B.suis OPS in 0.1 ml sterine saline as single dose per mouse. An exampleof a combination vaccine can bel ug each of B. abortus OPS+1 ug B.melitensis OPS-protein+1 ug B. suis OPS together in 0.1 ml sterilesaline as a single dose per mouse. Typically, for intra-peritoneal,sub-cutaneous and intra-muscular injections, the OPS were diluted in 0.1ml sterile saline, whereas for intra-nasal installation these were in0.01 ml (half given into each nostril). Unless otherwise specified, micewere allowed to rest for 5 weeks before challenge.

[0050] Oral administration of the vaccine was proven to be effective inthe case of B. abortus OPS vaccine to swine in Venezuela (U.S. Pat. No.5,951,987). While the Applicant did not test oral administration of thenew vaccine formulations of the present invention, it is suggested thatefficacy of these vaccines do extend to oral administration.

[0051] Challenges:

[0052] For intra-peritoneal challenge, a culture of Brucella was dilutedserially in 9 ml sterile saline blanks and 5×10⁵ bacteria (as confirmedby plating) in 0.1 ml of sterile saline was given to each mouse. Themice were allowed to rest for 1 week before being sacrificed andassessed. Past studies suggested that infection by intra-nasalinoculation was more difficult to take place (Cherwonogrodzky J. W., andDi Ninno, V. L. 1994, Brucella, brucellosis, undulant fever, AB—Is it athreat? A review in question and answer form (U), Suffield Memorandum1434, Defence Research Establishment Suffield, UNCLASSIFIED, page 6).Therefore, for intra-nasal challenge, 0.010 ml of a culture broth at anearly phase of growth (about 5×10⁷bacteria/0.010 ml) was used withoutdilution and the mice were allowed to rest for 2 weeks rather than 1week before being sacrificed and assessed.

[0053] Assessment of Infection:

[0054] Mice seldom show any symptoms when infected with Brucella,although with the more serious strains they may show ruffled, grayishlooking fur. Hence the only way to assess infection, was to weigh eachmouse, sacrifice these by cervical dislocation and remove organs such asspleens for weighing and obtaining bacterial counts. In this case, thespleens were weighed for the ratio spleen wt/body wt., then crushed in 1ml sterile saline by hand with a glass tissue grinder (Wheaton, 2 mlvolume). This suspension was removed, another 1 ml sterile saline wasadded to the chamber and crushing continued to complete the task and torinse the inside of the chamber. This second 1 ml, was pooled with thefirst. To prevent possible aerosol generation, the work was performedinside a Biosafety 2a or 2b Cabinet inside a Biocontainment Level 3(BL-3) area, and the investigator wore a seam sealed positive pressurehood (3M), HEPA filter with a blower powered by a battery pack, a sealedTyvek overall, double gloves and boots. Five tissue grinders were usedfor each group of mice and these were sterilized between groups. Eachtissue grinder had the chamber filled with 70% ethanol and the grindinghandle inserted therein. The chamber was topped up with 70% ethanol,sprayed with the ethanol to decontaminate the outside and then allowedto sit for 30 minutes. Thereafter, the ethanol was poured out, the topof the chamber and the grinding handle wiped with a KimWipe™ soaked in70% ethanol and the grinding handle allowed to air dry. The ethanol wasremoved from the chamber with a sterile pipette and the chamber rinsedwith sterile saline. Any adhering liquid was removed with anothersterile pipette. For the crushed spleen in 2 ml of saline noted above,0.1 ml of this was plated onto a plate of Brucella agar with 1.5 ppm ofcrystal violet, and 1 ml was transferred to a 9 ml sterile saline blankand dilutions with plating were repeated for these. Plates wereincubated for 2 hours at 37° C., 5% CO₂ and 90% humidity and the CFUcounted after one week incubation.

[0055] Separation of Blood Components:

[0056] In instances where only serum was required, mice were given adouble dose of 1:14 diluted Somnitol™ (pentabarbitol), in 0.5 ml permouse. Once anesthetized, a heart puncture was done with a 1 ml syringefitted with a 26-gauge needle, and whole blood was removed. Mice did notrecover from the amount of anaesthetic given. The blood was transferredto a 1.5 ml Eppendorf™ microcentrifuge tube and the tube was left in therefrigerator at 4° C. for a few hours to clot. It was then vortexedbriefly to loosen the clot and centrifuged at 2000× g for 5 minutes atroom temperature or at 22° C. to separate the serum, which formed thetop layer, from blood cells. The serum was removed with a Pasteurpipette, pooled with serum from other mice in the group, and filteredthrough a 0.2 um filter. It was used within a few hours of preparation.

[0057] To fractionate serum into different molecular weight groups,whole serum (about 10 ml from 20 vaccinated mice) was placed initiallyinto a 1000 molecular weight (m.w.) cutoff dialysis bag, and dialyzed ina 100 ml graduated cylinder against distilled water with magneticstirring within a 4° C. refrigerator. After 24 hours, the dialysate(1000 or less m.w. components), which remained in the cylinder, wasfrozen and freeze-dried. The serum was transferred to a 12,000 m.w.cutoff dialysis bag and dialyzed as before. This dialysate was1,000-12,000 m.w. and freeze-dried. The serum within the dialysis baghad 12,000 or greater m.w. components and was freeze-dried.

[0058] To separate blood components, initially 1 ml of sterile salinewas added to a 10 ml blood collection tube with heparin (10 foldheparin). One-tenth ml of 10-fold heparin was drawn into the 1 mlsyringe used to collect mouse blood. Whole blood (2 ml) was layered ontoan equal volume of Lymphoprep™ (Accurate Chemical and ScientificCorporation, Westbury, N.Y., USA) and centrifuged 1000× g for 30 minutesat room temperature. The top layer was serum which was drawn off with aPasteur pipette and then filtered through a 0.2 μm filter (final volumewas about 1.5 ml). Mononuclear and polymorphonuclear cells formed 2bands within the dextran solution. These were both drawn by a Pasteurpipette and washed twice with sterile saline (diluted in 0.85% sterilesaline) and centrifuged 2000× g for 30 min at room temperature. Thesupernatant layer was discarded and the cell pellet resuspended in 5 mlsterile saline, and washed as before and the cell pellet was resuspendedin 1 ml sterile saline. The red blood cell pellet was removed, washedwith sterile saline and resuspended in 1.5 ml of sterile saline.

[0059] The averages and standard error about the mean were calculatedusing the GraphPad Instat program (version 1.14).

[0060] Results and Discussion

EXAMPLE 1 Cross-Protection Study

[0061] In this study, female balb/c mice were injectedintra-peritoneally (i.p.) with either 0.1 ml sterile saline or B.abortus OPS vaccine/0.1 ml sterile saline. The mice were challenged 5weeks later with 5×10^(5(delete4)) bacteria/0.1 ml sterile saline. Theywere sacrificed and assessed one week later. The results are shown inTable 1. TABLE 1 Cross-protection study using a previous vaccineformulation Group of mice (5 mice/group) Spleen size Average number ofbacteria (CFU)/spleen Control: mice infected with B. abortus Normal13,200 ± 4,170  2308 Mice vaccinated with 1 ug OPS, infected Normal5,960 ± 2,440 with B. abortus 2308 Mice vaccinated with 100 ug OPS,infected Normal 164,000 ± 95,100  with B. abortus 2308 Control: miceinfected with B. suis 145 Large 4,460,000 ± 454,000   Mice vacinaed with1 ug OPS, infected Normal 908,000 ± 719,000 with B. suis 145 Micevaccinated with 100 ug OPS, infected Normal 511,000 ± 246,000 with B.suis 145 Control: mice infected with B. melitensis Large 279,000 ±36,800  16M Mice vaccinated with 1 ug OPS, infected Normal 325,000 ±183,000 with B. melitensis 16M Mice vaccinated with 100 ug OPS, infectedNormal 102,200 ± 18,000  with B. melitensis 16M

[0062] By convention, minimal protection is when vaccinates have 10-foldless bacteria in their spleens than unvaccinated controls. The aboveshows that the former vaccine formulation as disclosed in U.S. Pat. No.5,951,987 was not protective against B. melitensis 16M nor B. suis 145.It was also of little effect against B. abortus 2308, an unusual strainof B. abortus that sometimes changes its antigens to evade the immunesystem of the mouse (G. G. Schurig, personal communications, 1997). Asthe previous vaccine, formulated from B. abortus 1119-3 (that expressesthe “A” OPS) was not protective against B. melitensis 16M (thatexpresses “M” OPS), B. suis 145 (that expresses “M” as well as “A” OPS)nor in this case B. abortus 2308 (which is variable and sometimes shiftsfrom “A” to “M” OPS), it was apparent that the previous vaccine (asdisclosed in U.S. Pat. No. 5,951,987) was limited in its scope ofprotection. At the time of the previous patent submission, in which thevaccine formulated from B. abortus 1119-3 was protective against thespecies and strains of Brucella tested, this was unobvious. The previouspatent and past publication taught away from the new finding that acombination vaccine, one with more than one OPS, was needed for widerprotection against Brucella.

[0063] As noted in the above table, large doses of B. abortus 1119-3 OPS(i.e. 100 ug/mouse) did not offer any advantage for protection andindeed appeared to be counter-protective from B. abortus 2308 challenge.The Applicant has observed that low doses are more effective than highdoses, one dose is better than three doses, and formulations thatenhance antibody production (e.g. OPS on lipopolysaccharide, OPS inliposomes) are counter-productive. Antibody induction iscounter-productive because antibody will opsonize, or coat, invadingbacteria, these are recognized by white blood cells which ingest thebacterium, and then the parasitic bacterium is inside the host whiteblood cell, its preferred environment. In contrast, low doses of OPSvaccine appear to stimulate cell-mediated responses (Dr. John Wyckoff,Oklahoma State University, personal communications, 2000).

EXAMPLE 2 Effectiveness of Various Combination Vaccine Candidates onVaccinated Mice Infected One Day After Vaccination

[0064] TABLE 2 Response of vaccinated mice infected one day aftervaccination. Median bacterial numbers are shown in brackets. Each grouprepresents five mice. The animals were sacrificed seven days afterinfection. Vaccine given to group Spleen wt/body wt Bacterial CFU/spleenNone, saline control 0.0211 ± 0.0028 2,400,000 ± 250,000   (2,200,000) 1ug B. melitensis OPS 0.0163 ± 0.0027 1,690,000 ± 451,000   (1,860,000)100 ug B. melitensis OPS 0.0114 ± 0.0007 850,000 ± 361,000 (880,000) 0.5ug B. melitensis OPS + 0.5 ug B. abortus OPS 0.0172 ± 0.0012 1,820,000 ±381,000   (2,140,000) 50 ug B. melitensis OPS + 50 ug B. abortus 0.0129± 0.0015 984,000 ± 386,000 (680,000) 0.5 ug B. melitensis OPS-protein +0.5 ug B. 0.0089 ± 0.0003 624,000 ± 359,000 abortus OPS (96,000) 50 ugB. melitensis OPS-protein + 50 ug B. abortus 0.0183 ± 0.0015 1,550,000 ±351,000   OPS (1,560,000)

[0065] Throughout several years of studying Brucella infections in mice,it was evident that not all infected mice come down with brucellosis.Usually about 5-10% of the mice are infected, as evidenced by Brucellain their spleens, but as the numbers are trivial, they are obviously notdeveloping brucellosis. In the publication by Detilleux et al.(Detilleux, P. G., Deyoe, B. L., and Cheville, N. F. 1990. Penetrationand Intracellular Growth of Brucella abortus in nonphagocytic cells invitro. Infection and Immunity 58: 2320-2328) it was observed thatBrucella takes about 2 hours to infect mammalian cells. The Applicanthas also observed over the years that the most virulent forms ofBrucella (e.g. B. melitensis 16M) shed their OPS, potentiallyvaccinating the experimental animal before infection. Two questions wereasked. Does a combination vaccine protect against more strains andserotypes of Brucella? Does this vaccine work in a very short timeframe, such as a day instead of several weeks?

[0066] In the above data, initially it appears that none of the vaccineformulations were protective when given a day before infection. That isbecause a single mouse in a group of 5 that either was not responsive tothe vaccine or if the vaccine was not properly administered, will havehigh bacterial counts that will skew the average. As the data wasentered, it was obvious that the one highlighted (0.5 ug B. melitensisOPS-protein complex+0.5 ug B. abortus OPS) was actually very protective.In this instance, the median (or the middle number) was more reflectivethan the average bacterial count. It can also be seen that the vaccinedoes protect mice against B. melitensis 16M infection, even when givenas early as a day before infection (and other studies in the Applicant'slaboratory at the Defence Research Establishment Suffield (DRES) showedvaccine protection in as little as 1 hour before infection). Thepotential is that livestock being shipped to an area of high brucellosisprevalence, or a traveller or soldier travelling to a country whereBrucella is endemic, can be vaccinated with protection developing asthey are en route.

EXAMPLE 3 Effectiveness of Various Combination Vaccine Candidates onVaccinated Mice Infected Five Weeks After Vaccination

[0067] TABLE 3 Response of vaccinated mice infected five weeks aftervaccination. Median bacterial numbers are shown in brackets. Each grouprepresents five mice. The animals were sacrificed seven days afterinfection. Vaccine given to group Spleen wt/body wt Bacterial CFU/spleenNone, saline control 0.0117 ± 0.0015 1,120,000 ± 195,000   (1,300,000) 1ug B. melitensis OPS 0.0066 ± 0.0004 48,500 ± 23,000 (44,000) 100 ug B.melitensis OPS 0.0073 ± 0.0002 365,000 ± 167,000 (290,000) 0.5 ug B.melitensis OPS + 0.5 ug B. abortus OPS 0.0061 ± 0.0004 35,800 ± 6,460 (42,000) 50 ug B. melitensis OPS + 50 ug B. abortus 0.0065 ± 0.0005162,000 ± 70,000  (150,000) 0.5 ug B. melitensis OPS-protein + 0.5 ug B.0.0059 ± 0.0003 67,600 ± 30,200 abortus OPS (92,000) 50 ug B. melitensisOPS-protein + 50 ug B. abortus 0.0069 ± 0.0004 85,100 ± 26,000 OPS(70,000)

[0068] The results shown in Tables 2 and 3 above, demonstrate thatoverall, 0.5 ug B. melitensis OPS-protein+B. abortus OPS was mosteffective in providing protection against B. melitensis 16M infection,either one day or five weeks after vaccination. Higher dose of the samevaccine does not seem to be as effective, probably because it elicitantibodies. Tables 2 and 3 also show that given several weeks, OPSvaccines are effective for protecting mice from B. melitensis 16Minfections.

EXAMPLE 4 Effectiveness of Different Vaccine Candidates AgainstDifferent Species and Strains of Brucella Infections

[0069] In this case, Balb/c mice were vaccinated by the intra-peritonealroute with various vaccine candidates. The mice were challenged (i.p.)four weeks later with different species of Brucella. One week afterinfection, the mice were sacrificed and assessed for brucellosis. Eachgroup represents the average for five mice, each mouse was given 1 ug ofeach OPS. TABLE 4 Effect of various vaccines against different type ofBrucella infections. Spleen wt/body wt (first line) Bacterial count(CFU) in spleen (second and third line) B. melitensis 16M B. suis 145 B.abortus 30 B. abortus 2308 infection infection infection infectionControl - 0.0162 ± 0.0025 0.0052 ± 0.0008 0.0080 ± 0.0008 0.0047 ±0.0004 no vaccine 3,470,000 ± 740,000   346,000 ± 74,900  970,000 ±191,000 155,000 ± 74,300  B. abortus OPS 0.0077 ± 0.0012 0.0052 ± 0.00030.0077 ± 0.0014 0.0055 ± 0.0010 vaccine 270,000 ± 136,000 91,200 ±20,500 391,000 ± 146,000 7,740 ± 4,030 B. suis OPS vaccine 0.0055 ±0.0003 0.0045 ± 0.0003 0.0048 ± 0.0005 0.0050 ± 0.0005 6,650 ± 2,310 20± 11 148 ± 91  9,920 ± 5,090 B. melitensis OPS- 0.0059 ± 0.0006 0.0045 ±0.0004 0.0051 ± 0.0004 0,0065 ± 0.0008 protein 25,900 ± 13,800 0 ± 0 108± 59  238,000 ± 77,400  B. abortus OPS + B. 0.0055 ± 0.0004 0.0052 ±0.0005 0.0045 ± 0.0006 0.0053 ± 0.0005 melitensis OPS- 36,900 ± 16,40012 ± 5  212 ± 212 112,000 ± 63,300  protein B. abortus OPS + B. 0.0037 ±0.0002 0.0048 ± 0.0004 0.0050 ± 0.0003 0.0052 ± 0.0004 melitensis OPS-(no water 1 day) 1 ± 1 56 ± 51 84,900 ± 32,600 protein + B. suis OPS44,700 ± 31,400

[0070] The above results show that either a combination of OPS andOPS-protein antigens from different Brucellae, or an OPS preparationfrom a single strain of Brucella, (for example B. suis 145) thatexpresses more than one OPS, were effective in protecting mice frombrucellosis. The most remarkable of the vaccine “cocktails” tested isthat of B. suis 145 OPS. It not only protects against a very wide rangeof Brucella species, but it also appears to work the best for each. Itis believed that this greater OPS vaccine protection is because B. suis145 not only expresses both “A” and “M” (instead of just one) OPS, butthat it also expresses one (which Dr. Brad Berger at DRES has isolated)or more additional and previously unknown OPS.

[0071] It is also interesting to note that the vaccines work best (i.e.there is a greater contrast between non-vaccinated control mice andvaccinated mice) for the species and strains of Brucellae that are themost virulent (i.e. species or strains of Brucella that cause a largespleen sizes and high bacterial numbers in the spleens of unvaccinatedanimals). OPS is on the surface of smooth Brucella and it is also shedby the most virulent species and strains of Brucella. It is now believedthat the OPS is a virulence factor that reduces the resistance of thehost. By vaccinating with OPS and inducing an immunity against thiscomponent, it is likely that the host has an immunity both against theBrucella bacterium but also against the OPS that they shed.

EXAMPLE 5 Effectiveness of Different Vaccine Candidates Using VariousAdministration Routes

[0072] In this case, mice were vaccinated by different routes andvarious vaccines candidates were used. The mice were challenged fourweeks after vaccination with different species of Brucella and weresacrificed and assessed one week after vaccination. Each grouprepresents the average of 15 mice and each mouse was given 1 ug of OPS.TABLE 5 Effectiveness of different vaccine candidates using variousadministration of vaccine routes. Spleen wt/body wt Bacterial count(CFU) in spleen B. melitensis 16M B. suis 145 B. abortus 30 B. abortus2308 challenge, i.n. challenge, i.n. challenge, i.n. challenge, i.n.Saline control, i.m. 0.0082 ± 0.0004 0.0117 ± 0.0010 0.0047 ± 0.00010.0052 ± 0.0001 124,000 ± 34,900  448,000 ± 122,000 87 ± 24 7,040 ±2,540 B. suis OPS, i.n. 0.0088 ± 0.0004 288,000 ± 90,100  B. suis OPS,i.m. 0.0071 ± 0.0004 0.0082 ± 0.0006 0.0045 ± 0.0001 0.0048 ± 0.0001288,000 ± 90,100  48,500 ± 14,900 65 ± 27 954 ± 523 B. abortus OPS + B.0.0065 ± 0.0002 0.0067 ± 0.0005 0.0045 ± 0.0001 0.0050 ± 0.0003melitensis OPS- 22,000 ± 10,600 31,400 ± 11,600 20 ± 7  125 ± 68 protein + B. suis OPS, i.m. Saline control, s.c. 0.0074 ± 0.0004 0.01127± 0.0010  0.0049 ± 0.0001 0.0052 ± 0.0001 62,000 ± 21,900 339,000 ±94,400  230 ± 99  6,690 ± 2,040 B. suis OPS, s.c. 0.0060 ± 0.0005 0.0072± 0.0005 0.0046 ± 0.0002 0.0050 ± 0.0001 15,900 ± 12,000 29,000 ± 7,680 53 ± 19 361 ± 179 B. abortus OPS + B. 0.0064 ± 0.0002 0.0070 ± 0.00040.0047 ± 0.0001 0.0047 ± 0.0001 melitensis OPS- 7,100 ± 1,980 25,000 ±6,000  31 ± 20 154 ± 65  protein + B. suis OPS, s.c.

[0073] The above shows that either a “cocktail” vaccine, made by addingpurified antigens from different bacteria or by adding differentantigens prepared from one bacterium, given by different routes canprotect mice from a wide range of different Brucella species andstrains.

[0074] Recently the B. suis 145 OPS vaccine (1 ug/mouse) was given toanaesthetized mice (female, balb/c) by the intra-nasal route and micewere challenged 4 weeks later with B. suis 145 also given intra-nasally.Administration by this route did not appear to offer any protection. Atthe Applicant's research establishment, it has been observed thatvaccination against other infectious bacteria by the intranasal routeappears to induce antibodies in the respiratory tract (Dr. BillKournikakis, unpublished data). The induction of antibodies iscounter-productive for vaccine protection against Brucella. Recently ithas been found that B. suis 145 OPS offers protection in mice frombrucellosis when given in doses ranging from 1 ng to 100 ug. It was notprotective when doses were less than a nanogram. It is likely that thisvaccine can be effective for protecting mice from brucellosis when givenintranasally, but that the lower doses, in the nanogram rather thanmicrogram range, must be used to induce a cell-mediated response and toavoid a counter-productive antibody response.

EXAMPLE 6 Passive Immunity

[0075] Mice were vaccinated with 1 ug B. suis OPS intraperitoneally. Anhour later these were sacrificed and their serum collected. Half a ml ofserum was given to each naive mouse about 3 hours later. Recipient micewere challenged with B. melitensis 16M an hour after receiving the notedserum. The results are shown in Table 6. TABLE 6 Passive Immunity GroupMouse Mouse wt. (g) Spleen wt (g) Bacteria in spleen CONTROL - micereceived 0.5 ml of 1 23.52 0.4034 5,370,000 serum from unvaccinatedmice. 2 21.28 0.4682 4,280,000 Infected 1 hr later, assessed 1 week 322.24 0.4491 2,000,000 later 4 20.56 0.4027 1,860,000 5 23.36 0.46182,580,000 TEST - mice received 0.5 ml of serum 1 21.82 0.1174 620,000*from vaccinated mice. Infected 1 hr 2 19.52 0.4663 260,000* later,assessed 1 week later. 3 24.66 0.2162 1,000,000 4 21.64 0.6244 0 5 21.520.6765 2,360

[0076] The above shows that protection against brucellosis is very rapid(protection occurs within 1 hour of being vaccinated) and that thisprotection can be transferred by giving immune serum to naive mice togive them protection as well.

EXAMPLE 7 Passive Immunity (Continued)

[0077] In a second passive immunity study, mice were vaccinated with 1ug each with B. melitensis 16M OPS-protein given i.p, sacrificed fourweeks later, and their blood was fractionated. Naive mice (Table 7),were recipients (the number of mice used was small for this was proof ofconcept to justify further study). A control mouse was given salineintraperitoneally. For the other groups, these received (from the firstset of sacrificed mice) their red blood cells (0.5 ml/mouse), serum (0.5ml/mouse) or washed white blood cells (in 0.3 ml saline/mouse). Micegiven these injections were challenged one day later with 5×10⁴ bacteriagiven intra-peritoneally, and were sacrificed and assessed one weeklater. TABLE 7 Passive Immunity Mice were Body Spleen Bacteria in given:Mouse wt (g) wt. (g) spleen Saline 1 37.44 0.7547 5,340,000 Red bloodcells 1 37.52 0.4737 2,980,000 2 39.18 0.8639 2,840,000 3 37.12 0.75222,480,000 White blood cells 1 35.52 0.4611 740,000 2 36.86 0.39601,860,000 3 36.40 0.5360 1,000,000 Serum 1 39.66 0.4098 440,000 2 35.060.3956 2,500,000 3 37.92 0.2497 280,000

[0078] The above shows that in vaccinated mice, the protective factorappears to be made in white blood cells and then released into theserum. This protective factor is produced rapidly (within 1 hour asevidenced by Table 6), continues to be produced for several weeks (asevidenced by the above Table 7) and can be transferred to naive mice tooffer them protection from brucellosis as well.

We claim:
 1. A vaccine comprising a combination of outer-polysaccharidesextracted from Brucella, wherein said combination ofouter-polysaccharides comprises “A” outer-polysaccharide and “M”outer-polysaccharide and “R” protein antigens.
 2. A vaccine as claimedin claim 1, wherein said extracted outer-polysaccharide is at least 90percent pure.
 3. A vaccine as claimed in claim 1, wherein said “A”outer-polysaccharide is extracted from Brucella species selected fromthe group consisting of B. abortus biovar 1, B. abortus biovar 2, B.abortus biovar 3, B. abortus biovar 6, B. melitensis biovar 2, B. suisbiovar 1, B. suis biovar 2, B. suis biovar 3, B. neotomae and B. maris;said “M” outer-polysaccharide is extracted from Brucella speciesselected from the group consisting of B. abortus biovar 4, B. abortusbiovar 5, B. abortus biovar 9, B. melitensis biovar 1, B. suis biovar 5;and said “R” antigens are protein, core polysaccharide andouter-polysaccharide and are extracted from Brucella species selectedfrom the group consisting of B. ovis and B. canis.
 4. A vaccine asclaimed in claim 1, wherein said “A” and “M” outer-polysaccharides areextracted from Brucella species selected from the group consisting of B.abortus biovar 7, B. melitensis biovar 3 and B. suis biovar 4, and said“R” antigens are extracted from Brucella species selected from the groupconsisting of B. ovis and B. canis.
 5. A vaccine comprising acombination of outer-polysaccharides extracted from Brucella, whereinsaid combination comprises “A” outer-polysaccharide, “M”outer-polysaccharide and a Brucella outer-polysaccharide-proteincomplex.
 6. A vaccine as claimed in claim 5, wherein said extractedouter-polysaccharide is at least 90 percent pure.
 7. A vaccine asclaimed in claim 5, wherein said “A” outer-polysaccharide is extractedfrom Brucella species selected from the group consisting of B. abortusbiovar 1, B. abortus biovar 2, B. abortus biovar 3, B. abortus biovar 6,B. melitensis biovar 2, B. suis biovar 1, B. suis biovar 2, B. suisbiovar 3, B. neotomae and B. maris; said “M” outer-polysaccharide isextracted from Brucella species selected from the group consisting of B.abortus biovar 4, B. abortus biovar 5, B. abortus biovar 9, B.melitensis biovar 1, B. suis biovar 5; and saidouter-polysaccharide-protein complex is selected from the groupconsisting of outer-polysaccharide and Brucella membrane proteins,outer-polysaccharide and Brucella surface proteins, outer-polysaccharideand Brucella surface enzymes and outer-polysaccharide and Brucellacytoplasmic proteins.
 8. A vaccine as claimed in claim 5, wherein said“A” and “M” outer-polysaccharides are extracted from Brucella speciesselected from the group consisting of B. abortus biovar 7, B. melitensisbiovar 3 and B. suis biovar 4, and said outer-polysaccharide-proteincomplex is selected from the group consisting of outer-polysaccharideand Brucella membrane proteins, outer-polysaccharide and Brucellasurface proteins, outer-polysaccharide and Brucella surface enzymes andouter-polysaccharide and Brucella cytoplasmic proteins.
 9. A vaccine asclaimed in claim 1 or 5, wherein each of said outer-polysaccharide is inan amount of 1 ng to 100 ug for mice.
 10. A vaccine as claimed in claim9, wherein said outer-polysaccharide is in an amount of 1 ug for mice.11. A prophylactic method of protecting against brucellosis comprisingadministering to a mammal a vaccine as claimed in claims 1 to 10 priorto infection.
 12. A method as claimed in claim 11, wherein said vaccineis administered intra-peritoneally, sub-cutaneously, intra-muscularly,intra-nasally or orally.
 13. A prophylactic method of protecting againstbrucellosis comprising administering to a mammal, serum or white bloodcells obtained from mammals vaccinated with a vaccine as claimed inclaims 1 to 10.